title: Evidence on how a conserved glycine in the hinge region of HapR regulates its DNA binding ability: lessons from a natural variant.
abstract: HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapR(V2)) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapR(V2) to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapR(V2) (HapR(V2G)), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR(V2) and HapR(V2G) proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a "Y" shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.
authors.affiliation: Institute of Microbial Technology, Chandigarh, Council of Scientific and Industrial Research, Sector 39A, Chandigarh 160036, India.
authors.forename: Mitesh
authors.initials: M
authors.lastname: Dongre
authors.forename: Naorem Santa
authors.initials: NS
authors.lastname: Singh
authors.forename: Chetna
authors.initials: C
authors.lastname: Dureja
authors.forename: Nagesh
authors.initials: N
authors.lastname: Peddada
authors.forename: Ashish K
authors.initials: AK
authors.lastname: Solanki
authors.lastname: Ashish
authors.forename: Saumya
authors.initials: S
authors.lastname: Raychaudhuri
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D014644:Genetic Variation; D005998:Glycine / Q000502:physiology*; D017433:Protein Structure, Secondary / Q000502:physiology; D053038:Quorum Sensing / Q000235:genetics*; D014734:Vibrio cholerae / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004247:DNA; D005998:Glycine